The oncoproteins Mdm2 and its recently discovered family member Mdmx are frequently overexpressed in many human cancers. Both Mdm2 and Mdmx bind and are direct regulators of the p53 tumor suppressor, but their regulation of p53 activation is only partially understood. Moreover, Mdm2 and Mdmx also have other oncogenic functions that are not well characterized and that likely impact p53 activation and significantly contribute to tumorigenesis. Specifically, altered expression of either Mdm2 or Mdmx can lead to genome instability, which is a hallmark of cancer and facilitator of tumor development; however, the mechanisms for this remain unresolved and appear to have both p53-dependent and p53-independent components. Preliminary data suggest novel mechanisms of Mdm2 and Mdmx in the regulation of p53 and the DNA damage response. Therefore, we hypothesize that alterations in these functions of Mdm2 and Mdmx significantly contribute to their oncogenic activity that leads to genome instability and tumor development. We propose to investigate this hypothesis with three Specific Aims. Experiments in Aim 1 utilize innovative approaches to define the novel functions of Mdm2 and Mdmx that result in genome instability during tumorigenesis and the proteins and pathways required. Experiments in Aim 2 will evaluate a novel mechanism of p53 regulation by Mdm2 and Mdmx with multiple approaches, including a new mouse model. Experiments in Aim 3 will capitalize on the novel functions of Mdm2 and Mdmx in targeting an Achilles' heel in cancer cells, and will identify synthetic lethal combination for cells that have lost a functional Mdm2/Mdmx-p53 pathway. Results from these Aims will reveal critical insights into the oncogenic functions of Mdm2 and Mdmx and their roles in tumorigenesis. Our studies should also open new therapeutic avenues for the treatment of the 50% of human cancers that have inactivated p53.